Project Summary We have discovered that the predicted inactive pseudokinase selenoprotein O (SelO) adopts an atypical protein kinase fold, yet transfers AMP instead of phosphate to protein substrates in a post translational modification known as AMPylation. Our results illustrate the catalytic versatility of the protein kinase superfamily and suggest that AMPylation may be a more widespread post translational modification than previously appreciated. SelO localizes to the mitochondria, AMPylates proteins involved in cellular metabolism and redox biology, and appears to regulate an ancient and highly conserved cellular antioxidant signaling pathway. In higher eukaryotes, SelO contains the 21st genetically encoded amino acid, selenocysteine, which we propose functions as a redox sensor to regulate SelO activity in response to oxidative stress. Although reactive oxygen species are an obligatory part of human biology, elevated levels are characteristic of many disease states. For example, elevated reactive oxygen species can lead to DNA damage, which can initiate oncogenic transformation leading to cancer. Furthermore, alterations in redox homeostasis are implicated in the pathology of conditions such as stroke, heart attack, and peripheral vascular disease, all of which are major contributors to morbidity and mortality in United States. Therefore, a mechanistic understanding of the pathways that protect cells from oxidative stress could have major impacts on human health and disease. The major goal of this proposal is to determine the molecular mechanisms by which SelO-dependent AMPylation of mitochondrial proteins protects cells from oxidative stress and regulates redox homeostasis. As part of this work, we will determine the functional consequences of SelO-catalyzed AMPylation of a subset of substrates as well as the structural basis for the redox-dependent regulation of SelO activity. We anticipate that the results obtained herein will have the potential to define new paradigms of cellular regulation and redox signaling and could lead to innovative diagnostic tools or novel approaches for the treatment of human diseases.